Substituted chalcones as therapeutic compounds

ABSTRACT

This invention pertains to substituted chalcones, specifically substituted 1-(4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-ones, which have therapeutic application, for example, as potent antiproliferative agents and antiinflammatory agents, and which have the formula (I) wherein: X is —H, —OH, —OC(═O)R 3 , —OS(═O) 2 OH, or —OP(═O)(OH) 2 ; Y is —H or a C 1-4 alkyl group; Z is —H or —OCH 3 ; R 1  is —H, a C 1-4 alkyl group, or C 1-4 fluoroalkyl group; R 2  is —H, a C 1-4 alkyl group, or C 1-4 fluoroalkyl group; and, R 3  is —H, a C 1-6 alkyl group, a C 3-20 heterocyclyl group, or a C   5-20 aryl group; and pharmaceutically acceptable salts, esters, and protected forms thereof. The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, both in vitro and in vivo, for both diagnosis and treatment of, for example, proliferative conditions, such as cancer, and inflammatory conditions.

This application is the U.S. national phase of international applicationPCT/GB01/01341 filed 26 Mar. 2001, which designated the U.S.

RELATED APPLICATION

This application claims priority to United Kingdom (GB) PatentApplication No. 0007401.3, filed Mar. 27, 2000, which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

This invention pertains to substituted chalcones, specificallysubstituted 1-(4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-ones,which have therapeutic application, for example, as potentantiproliferative agents and antiinflammatory agents. The presentinvention also pertains to pharmaceutical compositions comprising suchcompounds, and the use of such compounds and compositions, both in vitroand in vivo, for both diagnosis and treatment of, for example,proliferative conditions, such as cancer, and inflammatory conditions.

BACKGROUND

Chalcone, also known as chalkone, benzylideneacetophenone,benzalacetophenone, and phenyl styryl ketone, is1,3-diphenyl-2-propen-1-one, and has the following structure:

A number of substituted chalcones have been prepared, with one or moresubstituents on the styryl phenyl group (left), the acyl phenyl group(right), and/or the double bond carbon atoms.

A number of substituted chalcones having a 3,4,5-trimethoxyphenyl group(as the acyl phenyl group) have been reported to have excellentantitumour activity (Hiromitsu, 1996; Ducki et al., 1998; Akihiko,1986). These compounds have the following general formula:

Surprisingly and unexpectedly, it has now been found that substitutedchalcones having a 3,5-dimethoxyphenyl group (as the acyl phenyl group)have highly potent anticancer activity and/or antiinflammatory activity.

One such compound, shown below (Chemical Abstracts Registry Number169803-62-7), has been reported (Berryman et al., 1995, 1997), but onlyas an intermediate used in the preparation of 5H-furan-2-one compounds,which are reported to have use as endothelin antagonists. Specifically,the compound shown below was prepared from 3,5-dimethoxyacetophenone and4-methoxybenzaldehyde (Example 122, page 146, in Berryman et al., 1995),subsequently derivatised (Examples 123 and 124, pages 147 and 148), andthen used as a reagent to prepare a number of 5H-furan-2-one compounds(Examples 125-135, pages 148-158).

SUMMARY OF THE INVENTION

One aspect of the present invention pertains to compounds of theformula:

wherein:

X is —H, —OH, —OC(═O)R³, —OS(═O)₂OH, or —OP(═O)(OH)₂;

Y is —H or a C₁₋₄alkyl group;

Z is —H or —OCH₃;

R¹ is —H, a C₁₋₄alkyl group, or C₁₋₄fluoroalkyl group;

R² is —H, a C₁₋₄alkyl group, or C₁₋₄fluoroalkyl group; and,

R³ is —H, a C₁₋₆alkyl group, a C₃₋₂₀heterocyclyl group, or

a C₅₋₂₀aryl group;

and pharmaceutically acceptable salts, esters, and protected formsthereof; with the proviso that X, Y, Z, R¹, and R² are not all —H.

In one preferred embodiment, X is —H.

In one preferred embodiment, X is —OH, —OC(═O)R³, —OS(═O)₂OH, or—OP(═O)(OH)₂.

In one preferred embodiment, X is —OH.

In one preferred embodiment, Y is —H. —CH₃ or —CH₂CH₃.

In one preferred embodiment, Y is —H.

In one preferred embodiment, Y is —CH₃ or —CH₂CH₃.

In one preferred embodiment, Z is —H.

In one preferred embodiment, Z is —OCH₃.

In one preferred embodiment, R¹ and R² are independently —H, —CH₃,—CH₂CH₃, —CF₃, —CH₂CF₃, or —CF₂CF₃.

In one preferred embodiment, both R¹ and R² are —H.

In one preferred embodiment, R³ is —CH₃. —CH₂CH₃, —C(CH₃)₃, or -Ph.

Another aspect of the present invention pertains to a compositioncomprising a compound as described herein (without the proviso) and apharmaceutically acceptable carrier.

Another aspect of the present invention pertains to a method of treatinga proliferative condition in a patient comprising administering to saidpatient a therapeutically-effective amount of a compound as describedherein (without the proviso). In one preferred embodiment, theproliferative condition is cancer.

Another aspect of the present invention pertains to a compound asdescribed herein (without the proviso), for use in a method of treatmentof the human or animal body.

Another aspect of the present invention pertains to use of a compound asdescribed herein (without the proviso) for the manufacture of amedicament for use in the treatment of a proliferative condition. In onepreferred embodiment, the proliferative condition is cancer.

Another aspect of the present invention pertains to a method ofprophylactically treating a proliferative condition in a patientcomprising administering to said patient a therapeutically-effectiveamount of a compound as described herein (without the proviso). In onepreferred embodiment, the proliferative condition is cancer.

Another aspect of the present invention pertains to the use of acompound as described herein (without the proviso) for the manufactureof a medicament for use in the prophylactic treatment of a proliferativecondition. In one preferred embodiment, the proliferative condition iscancer.

Another aspect of the present invention pertains to a method of treatinga inflammatory condition in a patient comprising administering to saidpatient a therapeutically-effective amount of a compound as describedherein (without the proviso). In one preferred embodiment, theinflammatory condition is rheumatoid arthritis, rheumatic fever,osteoarthritis, inflammatory bowel disease, psoriasis, or bronchialasthma.

Another aspect of the present invention pertains to the use of acompound as described herein (without the proviso) for the manufactureof medicament for use in the treatment of an inflammatory condition. Inone preferred embodiment, the inflammatory condition is rheumatoidarthritis, rheumatic fever, osteoarthritis, inflammatory bowel disease,psoriasis, or bronchial asthma.

Another aspect of the present invention pertains to a compound asdescribed herein (without the proviso), wherein X is —H, for use in amethod of diagnosis of the human or animal body. In one preferredembodiment, the diagnosis is for the presence of tumour cells expressingthe CYP1B1 enzyme.

Another aspect of the present invention pertains to the use of acompound as described herein (without the proviso), wherein X is —H, forthe presence of tumour cells expressing the CYP1B1 enzyme.

Another aspect of the present invention pertains to a method ofdiagnosis of a patient for the presence of tumour cells expressing theCYP1B1 enzyme, comprising:

(a) administering to the patient a compound as described herein (withoutthe proviso), wherein X is —H;

(b) determining the amount of the corresponding hydroxylated metabolite,wherein X is —OH, which is subsequently produced; and,

(c) correlating the amount with the presence or absence of the tumourcells in the patient.

As will be appreciated by one of skill in the art, features andpreferred embodiments of one aspect of the invention will also pertainto other aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of counts per minute (cpm) versus concentration ofCompound II (DMU-120) for the splenocyte anti-proliferation assaydescribed in Example 10.

DETAILED DESCRIPTION OF THE INVENTION

Compounds

One aspect of the present invention pertains to compounds of thefollowing formula:

wherein:

X is —H, —OH, —OC(═O)R³, —OS(═O)₂OH, or —OP(═O)(OH)₂;

Y is —H or a C₁₋₄alkyl group;

Z is —H or —OCH₃;

R¹ is —H, a C₁₋₄alkyl group, or C₁₋₄fluoroalkyl group;

R² is —H, a C₁₋₄alkyl group, or C₁₋₄fluoroalkyl group; and,

R³ is —H, a C₁₋₆alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀arylgroup;

and pharmaceutically acceptable salts, esters, and protected formsthereof.

Insofar as the present invention pertains to compounds, per se, thesecompounds are as defined herein, with the proviso that X, Y, Z, R¹, andR² are not all —H. However, this proviso does not apply to the presentinvention in its other aspects, for example, as it pertains topharmaceutical compositions comprising the compounds, methods oftreatment employing the compounds, the compounds for medical use, use ofthe compounds in the preparation of medicaments, and the like.

Note that the compounds of the present invention are all of the “E”(entgegen) or “trans” form, that is, the (optionally substituted)4-methoxy-phenyl group (styryl phenyl group) and the3,5-dimethoxybenzoyl group (acyl phenyl group) are positioned “trans”with respect to one another on the carbon-carbon double bond of theprop-1-ene backbone.

The term “C₁₋₄alkyl,” as used herein, pertains to monovalent aliphaticsaturated alkyl groups having from 1 to 4 carbon atoms. The term“aliphatic,” as used herein, pertains to groups which are linear orbranched, but not cyclic. Examples of saturated linear C₁₋₄alkyl groupsinclude methyl, ethyl, n-propyl, and n-butyl. Examples of saturatedbranched C₁₋₄alkyl groups include iso-propyl, iso-butyl, sec-butyl,tert-butyl. In one preferred embodiment, the C₁₋₄alkyl group is methylor ethyl.

The term “C₁₋₄fluoroalkyl group,” as used herein, pertains to aC₁₋₄alkyl group in which at least one hydrogen atom has been replacedwith a fluorine atom. Every hydrogen atom may be replaced with afluorine atom, in which case the group may conveniently be referred toas a “C₁₋₄perfluoroalkyl group.” Examples of C₁₋₄fluoroalkyl groupsinclude, but are not limited to, —CF₃, —CHF₂, —CH₂F, —CH₂CH₂F, —CH₂CHF₂,—CH₂CF₃, —CF₂CF₃, and —C(CF₃)₃. In one preferred embodiment, theC₁₋₄fluoroalkyl group is —CF₃, —CH₂CF₃, or —CF₂CF₃.

The term “C₁₋₆alkyl,” as used herein, pertains to monovalent alkylgroups having from 1 to 6 carbon atoms, which may be aliphatic oralicyclic, or a combination thereof. The term “aliphatic,” as usedherein, pertains to groups which are linear or branched, but not cyclic.The term “alicyclic,” as used herein, pertains to groups which have onering, or two or more rings (e.g., spiro, fused, bridged), but which arenot aromatic.

Examples of saturated linear C₁₋₆alkyl groups include, but are notlimited to, methyl, ethyl, n-propyl, n-butyl, and n-pentyl (amyl).

Examples of saturated branched C₁₋₆alkyl groups include, but are notlimited to, iso-propyl, iso-butyl, sec-butyl, tert-butyl, andneo-pentyl.

Examples of saturated alicylic (carbocyclic) C₁₋₆alkyl groups (alsoreferred to as “C₃₋₆cycloalkyl” groups) include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, as well as groupswhich comprise such groups, including, but not limited to,cyclopropylmethyl and cyclohexylmethyl.

The term “C₃₋₂₀heterocyclyl,” as used herein, pertains to a monovalentmoiety obtained by removing a hydrogen atom from a ring atom of analicyclic (i.e., non-aromatic cyclic) compound, said compound having onering, or two or more rings (e.g., spiro, fused, bridged), having from 3to 20 ring atoms, of which from 1 to 10 are ring heteroatoms, including,but not limited to, nitrogen, oxygen, and sulfur. Preferably, each ringhas from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.“C₃₋₂₀” denotes ring atoms, whether carbon atoms or heteroatoms.

Examples of C₃₋₂₀heterocyclyl groups having one nitrogen ring atominclude, but are not limited to, those derived from pyrrolidine,pyrroline, pyrrolinine, piperidine, dihydropyridine, andtetrahydropyridine.

Examples of C₃₋₂₀heterocyclyl groups having one oxygen ring atominclude, but are not limited to, those derived from oxolane(tetrahydrofuran), oxole (dihydrofuran), oxane (tetrahydropyran),dihydropyran, and pyran.

Examples of C₃₋₂₀heterocyclyl groups having one sulfur ring atominclude, but are not limited to, those derived from thiolane andtetrahydrothiopyran.

Examples of C₃₋₂₀heterocyclyl groups having two nitrogen ring atomsinclude, but are not limited to, those derived from imidazolidine,imidazoline, and piperazine.

Examples of C₃₋₂₀heterocyclyl groups having one nitrogen ring atom andone oxygen ring atom include, but are not limited to, those derived fromtetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole,dihydroiosoxazole, morpholine, tetrahydrooxazine, and dihydrooxazine.

Examples of C₃₋₂₀heterocyclyl groups having one oxygen ring atom and onesulfur ring atom include, but are not limited to, oxathiolane andoxathiane.

The term “C₅₋₂₀aryl,” as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from a ring atom of an aromaticcompound, said compound having one ring, or two or more fused rings, andhaving from 5 to 20 ring atoms. The ring atoms may be all carbon atoms,as in “carboaryl groups,” or may include one or more heteroatoms(including but not limited to oxygen, nitrogen, and sulfur), as in“heteroaryl groups.” In the latter case, the group may conveniently bereferred to as a “C₅₋₂₀heteroaryl” group, wherein “C₅₋₂₀” denotes ringatoms, whether carbon atoms or heteratoms. Preferably, each ring hasfrom 3 to 7 ring atoms, of which from 0 to 4 are ring heteroatoms.

Examples of C₅₋₂₀aryl groups which do not have heteroatoms (i.e.,carboaryl groups) include, but are not limited to, phenyl and naphthyl.

Examples of C₅₋₂₀heteroaryl groups include, but are not limited to,pyrrolyl, imidazolyl, pyrazolyl, triazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, triazinyl, furanyl, thienyl, thiazolyl,isothiazolyl, pyranyl, pyronyl, benzopyronyl, oxazolyl, isoxazolyl,oxadiazolyl, oxatriazolyl, oxathiazolyl, and oxathiazinyl.

The above C₁₋₆alkyl, C₃₋₂₀heterocyclyl, and C₅₋₂₀aryl groups maythemselves optionally be substituted with one or more substituents.Examples of such substituents include, but are not limited to,C₁₋₆alkyl, C₃₋₂₀heterocyclyl, and C₅₋₂₀aryl groups as well as halo,hydroxy, and carboxylic acid groups.

For example, the term “C₁₋₆alkyl-C₅₋₂₀aryl,” as used herein, describescertain C₅₋₂₀aryl groups which have been substituted with a C₁₋₆alkylgroup. Examples of such groups include, but are not limited to, tolyl,xylyl, mesityl, and cumenyl.

For example, the term “C₅₋₂₀aryl-C₁₋₆alkyl,” as used herein, describerscertain C₁₋₆alkyl groups which have been substituted with a C₅₋₂₀arylgroup. Examples of such groups include, but are not limited to, benzyl,tolylmethyl, and phenylethyl.

In one preferred embodiment, X is —H, and Y, Z, R¹, and R² are asdefined above. Such compounds may conveniently be referred to herein as“non-hydroxylated compounds.”

In one preferred embodiment, X is —OH, —OC(═O)R³, —OS(═O)₂OH, or—OP(═O)(OH)₂, and Y, Z, R¹, R², and R³ are as defined above.

In one preferred embodiment, X is —OH, and Y, Z, R¹, R², and R³ are asdefined above. Such compounds may conveniently be referred to herein as“hydroxylated compounds.”

In one preferred embodiment, X is —OC(═O)R³, —OS(═O)₂OH, or—OP(═O)(OH)₂, and Y, Z, R¹, R², and R³ are as defined above. Suchcompounds may conveniently be referred to herein as “esterifiedcompounds.”

In one preferred embodiment, Y is —H, —CH₃, or —CH₂CH₃, and X and Z areas defined above. In one preferred embodiment, Y is —H, and X and Z areas defined above.

In one preferred embodiment, Z is —H, and X and Y are as defined above.

In one preferred embodiment, R¹ and R² are independently —H, —CH₃,—CH₂CH₃, —CF₃, —CH₂CF₃, or —CF₂CF₃.

In one preferred embodiment, R¹ and R² are independently —H, —CH₃, or—CH₂CH₃.

In one preferred embodiment, R¹ and R² are independently —H, —CF₃,—CH₂CF₃, or —CF₂CF₃.

In one preferred embodiment, both R¹ and R² are —H.

In one preferred embodiment, only one of R¹ and R² is —H.

In one preferred embodiment, neither of R¹ and R² is —H.

In one preferred embodiment, one of R¹ and R² is —H, and the other is—CH₃, —CH₂CH₃, —CF₃, —CH₂CF₃, or —CF₂CF₃.

In one preferred embodiment, R³ is —CH₃ (so that —C(═O)R³ is —C(═O)CH₃,acetyl); —CH₂CH₃ (so that —C(═O)R³ is —C(═O)CH₂CH₃, propionyl); —C(CH₃)₃(so that —C(═O)R³ is —C(═O)C(CH₃)₃, pivaloyl); or -Ph (so that —C(═O)R³is —C(═O)Ph, benzoyl).

In one preferred embodiment:

X is —H, —OH, —OC(═O)R³, —OS(═O)₂OH, or —OP(═O)(OH)₂;

Y is —H, —CH₃ or —CH₂CH₃;

Z is —H or —OCH₃;

R¹ is —H, —CH₃, —CH₂CH₃, —CF₃, —CH₂CF₃, or —CF₂CF₃;

R² is —H, —CH₃, —CH₂CH₃, —CF₃, —CH₂CF₃, or —CF₂CF₃; and,

R³ is —H, —CH₃, —CH₂CH₃, —C(CH₃)₃, or -Ph

In one embodiment, the compound has the following structure, and isreferred to herein as Compound II (also referred to as DMU-120,(E)-1-(4-methoxyphenyl)-3-(3,5-dimethoxyphenyl) prop-1-en-3-one):

In one embodiment, the compound has the following structure, and isreferred to herein as Compound III (also referred to as DMU-153,(E)-1-(3-hydroxy-4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one):

In one embodiment, the compound has the following structure, and isreferred to herein as Compound IV (also referred to as DMU-162,(E)-1-(2,4-dimethoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one):

In one embodiment, the compound has the following structure, and isreferred to herein as Compound V (also referred to as DMU-436,(E)-2-(4-methoxyphenyl)-4-(3,5-dimethoxyphenyl)but-2-en-4-one):

In one embodiment, the compound has the following structure, and isreferred to herein as Compound VI (also referred to as DMU-428,(E)-1-(4-methoxyphenyl)-2-methyl-3-(3,5-dimethoxyphenyl)prop-1-en-3-one):

In one embodiment, the compound has the following structure, and isreferred to herein as Compound VII (also referred to as DMU-153,(E)-1-(3—Hydroxy-4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-oneoxime), wherein the ketone group is protected in the form of a Schiffbase:

In one embodiment, the compound has the following structure, and isreferred to herein as Compound VIII (also referred to as DMU-170,(E)-1-(3-acetoxy-4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one):

In one embodiment, the compound has the following structure((E)-1-(3-methyl-4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one):

In one embodiment, the compound has the following structure((E)-1-(3-hydroxy-4-methoxy-5-methylphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one):

In one embodiment, the compound has the following structure((E)-1-(2,4-dimethoxy-5-hydroxy-phenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one):

In one embodiment, the compound has the following structure((E)-1-(2,4-dimethoxy-3-methyl-phenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one):

In one embodiment, the compound has the following structure((E)-1-(2,4-dimethoxy-3-methyl-4-hydroxy-phenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one):

Isomers, Salts, Hydrates, Protected Forms, and Prodrugs

A certain compound may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, stereoisomeric,tautomeric, conformational, or anomeric forms, including but not limitedto, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo-and exo-forms; R-, S-, and meso-forms; D- and L-forms; (+) and (−)forms; keto- and enol-forms; syn- and anti-forms; synclinal- andanticlinal-forms; α- and β-forms; axial and equatorial forms; boat-,chair-, twist-, envelope-, and halfchair-forms; and combinationsthereof, hereinafter collectively referred to as “isomers” (or “isomericforms”). Note that specifically excluded from the terms “isomers,” asused herein, are structural (or constitutional) isomers (i.e., isomerswhich differ in the connections between atoms rather than merely by theposition of atoms in space). For example, a reference to a methoxygroup, —OCH₃, is not to be construed as a reference to its structuralisomer, a hydroxymethyl group, —CH₂OH. However, a reference to a classof structures may well include structurally isomeric forms fallingwithin that class (e.g., C₁₋₆alkyl includes n-propyl and iso-propyl;butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includesortho-, meta-, and para-methoxyphenyl).

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including racemic and other mixturesthereof. Methods for the preparation (e.g., asymmetric synthesis) andseparation (e.g., fractional crystallisation and chromatographic means)of such isomeric forms are either known in the art or are readilyobtained by adapting the methods taught herein in a known manner.

As noted above, the compounds of the present invention are all of the“E” (entgegen) or “trans” form, that is, the (optionally substituted)4-methoxy-phenyl group (styryl phenyl group) and the3,5-dimethoxybenzoyl group (acyl phenyl group) are positioned “trans”with respect to one another on the carbon-carbon double bond of theprop-1-ene backbone.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977.

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g., —COOH may be —COO⁻; —OSO₃H may be —OSO₃ ⁻; —OP(═O)(OH)₂ may be —OP(═O) (OH)O⁻ or —OP(═O) (O⁻)₂), then a salt may be formedwith a suitable cation.

Examples of suitable inorganic cations include, but are not limited to,alkali metal ions such as Na+ and K+, alkaline earth cations such asCa²⁺ and Mg²⁺, and other cations such as Al⁺³. Examples of suitableorganic cations include, but are not limited to, ammonium ion (i.e., NH₄⁺) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺).Examples of some suitable substituted ammonium ions are those derivedfrom: ethylamine, diethylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine. An example of a common quaternary ammoniumion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g., —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous. Examples of suitable organicanions include, but are not limited to, anions from the followingorganic acids: acetic, trifluoroacetic, propionic, isobutyric, succinic,gycolic, suberic, sebacic, stearic, caprylic, lactic, malic, tartaric,citric, ascorbic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, chlorobenzoic, methylbenzoic, dinitrobenzoic, phthalic,salicylic, sulfanilic, 2-acetyoxybenzoic, fumaric, benzenesulfonic,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and valeric.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding hydrate of the active compound, for example, as themono-hydrate, the di-hydrate, the tri-hydrate, etc.

It may be convenient or desirable to prepare, purify, and/or handle theactive compound in a chemically protected form. The term “chemicallyprotected form,” as used herein, pertains to a compound in which one ormore reactive functional groups are protected from undesirable chemicalreactions, that is, are in the form of a protected or protecting group(also known as a masked or masking group and a blocked or blockinggroup).

For example, a hydroxy group may be protected as an ether (—OR) or anester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl(diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl ort-butyldimethylsilyl ether; or an acetyl ester (—OC(═O)CH₃, —OAc).

For example, an aldehyde or ketone group may be protected as an acetalor ketal, respectively, in which the carbonyl group (>C═O) is convertedto a diether (>C(OR)₂), by reaction with, for example, a primaryalcohol. The aldehyde or ketone group is readily regenerated byhydrolysis using a large excess of water in the presence of acid. Forexample, the ketone group of the present compounds may be protected inthe form of a Schiff Base, by condenation with a suitable amine, or asan oxime, by reaction with hydroxylamine.

It may be convenient or desirable to prepare, purify, and/or handle theactive compound in the form of a prodrug. The term “prodrug,” as usedherein, pertains to a compound which, when metabolised, yields thedesired active compound. Typically, the prodrug is inactive, or lessactive than the active compound, but may provide advantageous handling,administration, or metabolic properties. For example, some prodrugs areesters of the active compound; during metabolism, the ester group iscleaved to yield the active drug. Also, some prodrugs are activatedenzymatically to yield the active compound, or a compound which, uponfurther chemical reaction, yields the active compound. For example, theprodrug may be a sugar derivative or other glycoside conjugate, or maybe an amino acid ester derivative.

Synthesis

The compounds of the present invention may be prepared, for example, byAldol condensation of the corresponding carbonyl compounds A and B, asillustrated below in Scheme 1.

For example, compound III may be prepared by stirring a solution of3-hydroxy-4-methoxybenzaldehyde and 3,5-dimethoxyacetophenone inmethanol (as solvent) with added aqueous sodium hydroxide solution (asbase catalyst) for 18 hours at ambient temperature. The mixture isacidified with hydrochloric acid to give the product which is thencollected by filtration. The reaction is illustrated below in Scheme 2.

Additional methods for the preparation of compounds of the presentinvention, for example, where R¹ and/or R² are other than hydrogen, aredescribed in the Examples below.

Compounds for which X is —OC(═O)R³, —OS(═O)₂OH, or —OP(═O)(OH)₂ may beprepared from their hydroxy analogs (where X is —OH) by reaction with anorganic acid (i.e., R³COOH) or an inorganic acid (i.e., sulfuric acid,H₂SO₄; phosphoric acid, H₃PO₄).

The groups —OS(═O)₂OH and —OP(═O)(OH)₂ may be present as such, in theirfree acid form, or they may be present as a salt or ester thereof, asdiscussed above. For example, the group —OS(═O)₂OH may be present as—OS(═O)₂O⁻ M⁺, wherein M⁺ is a suitable cation. Similarly, the group—OP(═O)(OH)₂ may be present as —OP(═O)(OH)O⁻ M⁺ or —OP(═O)(O⁻)₂(M⁺)₂,wherein M⁺ is a suitable cation. Examples of suitable cations arediscussed above. In one embodiment, the group —OP(═O)(OH)₂ is present asthe disodium salt, —OP(═O)(O⁻)₂(Na⁺)₂. Other salts and esters aredescribed in Pettit et al, 1995.

Active Compounds and Prodrugs of Active Compounds

As demonstrated in the examples below, the compounds of the presentinvention are highly potent antiproliferative agents, and/or areprodrugs for highly potent antiproliferative agents.

Compounds of the present invention which exhibit low or moderateintrinsic activity may act as prodrugs, and be metabolically activatedto generate more potent compounds of the present invention. This isespecially useful in cancer therapy where metabolic activation can beachieved by an enzyme that is expressed in tumours. For example, thecytochrome P-450 enzyme CYP1B1 has been shown to be specificallyexpressed in tumour cells, but is not found in the corresponding normaltissues. This enzyme is found to be expressed in a variety of tumours,such as brain, breast, colon, stomach, ovarian and prostate cancers(Murray et al, 1997).

For example, compound III is a very potent anticancer agent withactivity against a number of human tumour cells lines. Compound II, haslow intrinsic activity, but is metabolised by CYP1B1 through an aromatichydroxylation reaction to generate the potent anticancer agent compoundIII. In this way, compound II may act as a non-toxic prodrug which isactivated by CYP1B1 to generate the highly potent anticancer compound offormula III, as illustrated in Scheme 3, below.

In such cases, the prodrug is useful as a selective anticancer agentwith low intrinsic toxicity. Furthermore, prodrugs with low intrinsiccytotoxicity, which are only activated upon entering tumour cellscontaining the CYP1B1enzyme, are not only useful for treating cancer,but also as a prophylactic, in cancer prevention (i.e., as a cancerpreventative agent).

Furthermore, the hydroxylated metabolite, compound III, exhibits muchgreater fluorescence than the prodrug, compound II, and this propertymay be exploited in the diagnosis of cancer, by detecting and/ormeasuring the formation of the hydroxylated metabolite.

Thus, one aspect of the present invention pertains to a method ofdiagnosis of a patient for the presence of tumour cells expressing theCYP1B1 enzyme, comprising:

(a) administering to the patient a non-hydroxylated prodrug as describedherein, wherein X is —H (e.g., compound II);

(b) determining the amount of the corresponding hydroxylated metabolite,wherein X is —OH (e.g., compound III) which is subsequently produced;and,

(c) correlating the amount with the presence or absence of the tumourcells in the patient.

Use of Compounds

The present invention provides active compounds, specifically, activesubstituted-1-(4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-ones, which regulate cell proliferation, as well as methodsof regulating cell proliferation, comprising contacting a cell with aneffective amount of an active compound, whether in vitro or in vivo.

The term “active,” as used herein, pertains to compounds which arecapable of regulating cell proliferation, and specifically includes bothcompounds with intrinsic activity (drugs) as well as prodrugs of suchcompounds, which prodrugs may themselves exhibit little or no intrinsicactivity.

One of ordinary skill in the art is readily able to determine whether ornot a candidate compound is active, that is, regulates cellproliferation for any particular cell line. For example, one assay whichmay conveniently be used to assess the proliferation regulation offeredby a particular compound is described in the examples below.

For example, a sample of cells (e.g., from a tumour) may be grown invitro and a candidate compound brought into contact with the cells, andthe effect of the compound on those cells observed. As examples of“effect,” the morphological status of the cells may be determined (e.g.,alive or dead), or the expression levels of genes associated with cellcycle regulation determined. Where the candidate compound is found toexert an influence on the cells, this may be used as a prognostic ordiagnostic marker of the efficacy of the compound in methods of treatinga patient carrying the tumour or a tumour of the same cellular type.

Thus, in one aspect, the present invention provides antiproliferativeagents. The term “antiproliferative agent” as used herein, pertain to acompound which treats a proliferative condition (i.e., a compound whichis useful in the treatment of a proliferative condition).

The terms “cell proliferation,” “proliferative condition,”“proliferative disorder,” and “proliferative disease,” are usedinterchangeably herein and pertain to an unwanted or uncontrolledcellular proliferation of excessive or abnormal cells which isundesired, such as, neoplastic or hyperplastic growth, whether in vitroor in vivo. Examples of proliferative conditions include, but are notlimited to, pre-malignant and malignant cellular proliferation,including but not limited to, malignant neoplasms and tumours, cancers,leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g.,of connective tissues), and atherosclerosis. Any type of cell may betreated, including but not limited to, lung, colon, breast, ovarian,prostate, liver, pancreas, brain, and skin.

Antiproliferative compounds of the present invention have application inthe treatment of cancer, and so the present invention further providesanticancer agents. The term “anticancer agent” as used herein, pertainsto a compound which treats a cancer (i.e., a compound which is useful inthe treatment of a cancer). The anti-cancer effect may arise through oneor more mechanisms, including but not limited to, the regulation of cellproliferation, the inhibition of angiogenesis (the formation of newblood vessels), the inhibition of metastasis (the spread of a tumourfrom its origin), the inhibition of invasion (the spread of tumour cellsinto neighbouring normal structures), or the promotion of apoptosis(programmed cell death).

The present invention also provides active compounds, specifically,active substituted-1-(4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-ones, which are useful in the treatment of inflammatoryconditions. For example, such compounds have growth down-regulatoryeffects on splenocytes. Examples of inflammaotry conditions include, butare not limited to, rheumatoid arthritis, rheumatic fever,osteoarthritis, inflammatory bowel disease, psoriasis, and bronchialasthma.

The invention further provides active compounds for use in a method oftreatment of the human or animal body. Such a method may compriseadministering to such a subject a therapeutically-effective amount of anactive compound, preferably in the form of a pharmaceutical composition.

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, amelioration of the condition,and cure of the condition. Treatment as a prophylactic measure is alsoincluded.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of an active compound, or a material, composition or dosagefrom comprising an active compound, which is effective for producingsome desired therapeutic effect, commensurate with a reasonablebenefit/risk ratio.

The invention further provides the use of an active compound for themanufacture of a medicament, for example, for the treatment of aproliferative condition or an inflammatory condition, as discussedabove.

The invention further provides a method of treatment of the human oranimal body, the method comprising administering to a subject in need oftreatment a therapeutically-effective amount of an active compound,preferably in the form of a pharmaceutical composition.

Active compounds may also be used as part of an in vitro assay, forexample, in order to determine whether a candidate host is likely tobenefit from treatment with the compound in question.

Active compounds may also be used as a standard, for example, in anassay, in order to identify other active compounds, otherantiproliferative agents, other antiinflammatory agents, etc.

Administration

The active compound or pharmaceutical composition comprising the activecompound may be administered to a subject by any convenient route ofadministration, whether systemically/peripherally or at the site ofdesired action, including but not limited to, oral (e.g., by ingestion);topical (including transdermal, intranasal, ocular, buccal, andsublingual); pulmonary (e.g., by inhalation therapy using, for example,an aerosol); rectal; vaginal; parenteral, for example, by injection,including subcutaneous, intradermal, intramuscular, intravenous,intraarterial, intracardiac, intrathecal, intraspinal, intracapsular,subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular,intraarticular, subarachnoid, and intrasternal.

The subject may be a eukaryote, an animal, a vertebrate animal, amammal, a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine(e.g., a mouse), a simian (e.g., a chimpanzee), or a human.

Formulations

While it is possible for the active ingredient to be administered alone,it is preferable to present it as a pharmaceutical composition (e.g.,formulation) comprising at least one active ingredient, as definedabove, together with one or more pharmaceutically acceptable carriers,excipients, diluents, buffers, adjuvants, stabilisers, or othermaterials well known to those skilled in the art and optionally othertherapeutic agents.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgement, suitable for use in contactwith the tissues of a subject (e.g., human) without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Each carrier,excipient, etc. must also be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Suchmethods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then if necessaryshaping the product.

Formulations may be in the form of liquids, solutions, suspensions,emulsions, tablets, losenges, granules, powders, capsules, cachets,pills, ampoules, suppositories, pessaries, ointments, gels, pastes,creams, sprays, foams, lotions, oils, boluses, electuaries, or aerosols.

Formulations suitable for oral administration (e.g., by ingestion) maybe presented as discrete units such as capsules, cachets or tablets,each containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion; as a bolus; as an electuary; or as apaste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g., sodiumstarch glycolate, cross-linked povidone, cross-linked sodiumcarboxymethyl cellulose), surface-active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile. Tablets may optionally beprovided with an enteric coating, to provide release in parts of the gutother than the stomach.

Formulations suitable for topical administration (e.g., transdermal,intranasal, ocular, buccal, and sublingual) may be formulated as anointment, cream, suspension, lotion, powder, solution, past, gel, spray,aerosol, or oil. Alternatively, a formulation may comprise a patch or adressing such as a bandage or adhesive plaster impregnated with activeingredients and optionally one or more excipients or diluents.

Formulations suitable for topical administration in the mouth includelosenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of about 20 to about 500 microns which is administered in themanner in which snuff is taken, i.e., by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid for administrationas, for example, nasal spray, nasal drops, or by aerosol administrationby nebuliser, include aqueous or oily solutions of the activeingredient.

Formulations suitable for topical administration via the skin includeointments, creams, and emulsions. When formulated in an ointment, theactive ingredient may optionally be employed with either a paraffinic ora water-miscible ointment base. Alternatively, the active ingredientsmay be formulated in a cream with an oil-in-water cream base. Ifdesired, the aqueous phase of the cream base may include, for example,at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol havingtwo or more hydroxyl groups such as propylene glycol, butane-1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol and mixturesthereof. The topical formulations may desirably include a compound whichenhances absorption or penetration of the active ingredient through theskin or other affected areas. Examples of such dermal penetrationenhancers include dimethylsulfoxide and related analogues.

When formulated as a topical emulsion, the oily phase may optionallycomprise merely an emulsifier (otherwise known as an emulgent), or itmay comprises a mixture of at lease one emulsifier with a fat or an oilor with both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabiliser. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabiliser(s) make up theso-called emulsifying wax, and the wax together with the oil and/or fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80,cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodiumlauryl sulphate. The choice of suitable oils or fats for the formulationis based on achieving the desired cosmetic properties, since thesolubility of the active compound in most oils likely to be used inpharmaceutical emulsion formulations may be very low. Thus the creamshould preferably be a non-greasy, non-staining and washable productwith suitable consistency to avoid leakage from tubes or othercontainers. Straight or branched chain, mono- or dibasic alkyl esterssuch as di-isoadipate, isocetyl stearate, propylene glycol diester ofcoconut fatty acids, isopropyl myristate, decyl oleate, isopropylpalmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branchedchain esters known as Crodamol CAP may be used, the last three beingpreferred esters. These may be used alone or in combination depending onthe properties required. Alternatively, high melting point lipids suchas white soft paraffin and/or liquid paraffin or other mineral oils canbe used.

Formulations suitable for rectal administration may be presented as asuppository with a suitable base comprising, for example, cocoa butteror a salicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient, such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration (e.g., by injection,including cutaneous, subcutaneous, intramuscular, intravenous andintradermal), include aqueous and non-aqueous isotonic, pyrogen-free,sterile injection solutions which may contain anti-oxidants, buffers,preservatives, stabilisers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. Examples of suitable isotonic vehicles for use insuch formulations include Sodium Chloride Injection, Ringer's Solution,or Lactated Ringer's Injection. Typically, the concentration of theactive ingredient in the solution is from about 1 ng/ml to about 10μg/ml, for example from about 10 ng/ml to about 1 μg/ml. Theformulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampoules and vials, and may be stored in afreeze-dried (lyophilised) condition requiring only the addition of thesterile liquid carrier, for example water for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules, and tablets. Formulations maybe in the form of liposomes or other microparticulate systems which aredesigned to target the active compound to blood components or one ormore organs.

Dosage

It will be appreciated that appropriate dosages of the active compounds,and compositions comprising the active compounds, can vary from patientto patient. Determining the optimal dosage will generally involve thebalancing of the level of therapeutic benefit against any risk ordeleterious side effects of the treatments of the present invention. Theselected dosage level will depend on a variety of factors including, butnot limited to, the activity of the particular compound, the route ofadministration, the time of administration, the rate of excretion of thecompound, the duration of the treatment, other drugs, compounds, and/ormaterials used in combination, and the age, sex, weight, condition,general health, and prior medical history of the patient. The amount ofcompound and route of administration will ultimately be at thediscretion of the physician, although generally the dosage will be toachieve local concentrations at the site of action which achieve thedesired effect.

Administration in vivo can be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration arewell known to those of skill in the art and will vary with theformulation used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician.

In general, a suitable dose of the active compound is in the range ofabout 0.01 to about 100 mg per kilogram body weight of the subject perday. Where the active ingredient is a salt, an ester, prodrug, or thelike, the amount administered is calculated on the basis the parentcompound and so the actual weight to be used is increasedproportionately.

EXAMPLES

The following are examples are provided solely to illustrate the presentinvention and are not intended to limit the scope of the invention, asdescribed herein. Unless otherwise specified, all reagents werepurchased from Sigma-Aldrich, Dorset, UK.

Example 1 (E)-1-(4-Methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one(Compound II, DMU-120)

To a stirred solution of 4-methoxybenzaldehyde (0.709 g, 5.21 mmol) and3,5-dimethoxyacetophenone (0.939 g, 5.21 mmol) in methanol (20 ml) wasadded a 50% w/v solution of aqueous NaOH (4 ml). The mixture was stirredfor 1 h at room temperature. The mixture was acidified (conc. HCl) andthe resultant precipitate collected by filtration. Recrystallisationfrom methanol afforded the product as fine pale yellow crystals (1.05 g,68%).

Mp 87-88° C.; ¹H NMR δ (CDCl₃) 3.85 (6H, s, OCH₃), 3.86 (3H, s, OCH₃),6.65 (1H, s), 6.95 (2H, d), 7.15 (2H, s), 7.35 (1H, d), 7.60 (2H, d),7.75 (1H, d); ¹³C NMR d (CDCl₃) 55.42, 55.62, 104.78, 106.25, 114.41,119.75, 127.56, 130.26, 140.51, 144.84, 160.83, 161.69, 190.18; MS (relintensity) m/z 299 ([M+H]⁺, 100%); IR ν_(max) (KBr)/cm⁻¹ 1650 (C═O).

Example 2(E)-1-(3-Hydroxy-4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one(Compound III, DMU-153)

To a stirred solution of 3-hydroxy-4-methoxybenzaldehyde (0.76 g, 5mmol) and 3,5-dimethoxyacetophenone (0.90 g, 5 mmol) in methanol (10 ml)was added aqueous NaOH (4 ml, 50% w/v) and the mixture stirred for 18 hat room temperature. The mixture was acidified (conc. HCl, 6 ml) and theresultant precipitate collected by filtration. The crude solid wasdissolved in chloroform, washed with water (2×50 ml), and dried overanhydrous sodium sulfate. Evaporation and recrystallisation frommethanol afforded the product as a pale yellow solid (0.75 g, 48%).

Mp 129° C.; IR ν_(max) (KBr)/cm⁻¹ 1660 (C═O). ¹H NMR δ (CDCl₃) 3.86 (6H,s, OCH₃), 3.93 (3H, s, OCH₃), 5.73 (1H, s, OH), 6.66 (1H, t, J=2.3 Hz,H-4′), 6.65 (1H, d, J=8.35 Hz, H-5), 7.13 (3H, m, ArH), 7.27 (1H, d,J=2.1 Hz, H-2), 7.33 (1H, d, J=15.6 Hz, CH), 7.73 (1H, d, J=15.6 Hz,CHCO); ¹³C NMR d (CDCl₃) 55.67, 56.07, 105.03, 106.28, 110.62, 113.09,120.31, 122.80, 128.56, 140.50, 144.97, 145.96, 148.91, 160.91, 190.15;MS (rel intensity) m/z 315 ([M+H]⁺, 100%); Anal. Calcd(C₁₈H₁₈O₅.0.5H₂O): C, 66.86; H, 5.92. Found C, 66.83; H, 5.91.

Example 3(E)-1-(2,4-dimethoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one(Compound IV, DMU-162)

A mixture of 2,4-dimethoxybenzaldehyde (1.89 g, 0.011 mol),3,5-dimethoxyacetophenone (2.05 g, 0.011 mol) and 50% w/v of aqueoussodium hydroxide (9.1 ml, 0.11 mol) in methanol (20 ml) were stirred atroom temperature for 1 hour. The yellow solid that precipitated wasfiltered and sequentially washed with cold methanol and ether andfinally dried in a vacuum dessicator. Purification by recrystallisationfrom methanol gave 2.7 g (73%) of the product as yellow crystals.

¹H-NMR (CDCl₃) 8.0 (d, 1H), 7.5 (d, 1H), 7.4 (d, 1H), 7.1 (d, 2H), 6.65(m, 1H), 6.5 (dd, 1H), 6.4 (d, 1H), 3.85 (s, 3H), 3.8 (s, 3H), 3.75 (s,6H). ¹³C NMR (CDCl₃) 190.6, 163.1, 160.8, 160.4, 140.8, 140.5, 130.8,121.5, 120.3, 117.0, 107.5, 106.2, 105.0, 98.4, 56.6, 55.5, 54.3. MassSpectrum m/e (M+1) 329.

3,5-Dimethoxyacetophenone trimethylsilyl enol ether was prepared byreaction of 3,5-dimethoxyacetophenone withtrimethylsilyltrifluoromethanesulfonate in the presence oftrimethylamine base, in dichloromethane as solvent. A dichloromethanesolution was prepared containing 4-methoxyacetophenone (0.3 g, 2 mmol),3,5-dimethoxy-acetophenone trimethylsilyl enol ether (2 mmol) andtriethylamine (0.55 ml, 4 mmol). To this solution was addedtrifluoroacetic anhydride (0.28 ml, 2 mmol), followed by titaniumtetrachloride (0.38 g, 2 mmol), and the resulting mixture stirred atambient temperature for 4 hours. The product was purified by columnchromatography (SiO₂, petroleum:ether (40:60 v/v) with an increasinggradient elution of ethyl acetate, 0-25%) to furnish the product as acolourless oil. Mass Spectrum m/e (M+1) 313.

Example 5(E)-1-(4-methoxyphenyl)-2-methyl-3-(3,5-dimethoxyphenyl)prop-1-en-3-one(Compound VI, DMU-428)

(a) 3,5-Dimethoxybenzaldehyde (1 g, 6 mmol), in dry tetrahydrofuran (20ml), was added over 15 min to ethylmagnesium bromide (7.23 ml (1.0 Msolution in tetrahydrofuran), 7.2 mmol) in dry tetrahydrofuran (10 ml)at 0° C., under nitrogen. After refluxing the mixture for 18 hours, agrey solution was obtained. The reaction was then quenched by adding iceand 1 M hydrochloric acid (20 ml) dropwise and the aqueous phase wasextracted with ether (3×25 ml), the combined organic layers were driedover anhydrous magnesium sulfate and reduced in vacuo. Purification bycolumn chromatography (SiO₂, petroleum:ether (40:60 v/v) with anincreasing gradient of ethyl acetate, 0-20%) gave 0.85 g (72%) of thealcohol as a yellow oil.

(b) To a stirred solution of dimethylsulfoxide (0.694 ml, 9.8 mmol) indry dichloromethane (5 ml) at −78° C. was added, over 15 min, oxalylchloride (0.424 ml, 4.9 mmol) under nitrogen. The solution was stirredfor 15 min at −78° C. until the evolution of gas stopped, then asolution of the alcohol (0.85 g, 4.3 mmol) in dichloromethane (5 ml) wasadded over 15 min. The mixture was stirred at −78° C. for a further 30min before triethylamine (3.0 ml, 21.6 mmol) was added over 10 min, thiswas stirred for a further 5 min at −78° C. and then allowed to warm upto room temperature and left for 2 hours. The mixture was then dilutedwith dichloromethane (20 ml) and the organic layer was sequentiallywashed with 1 M hydrochloric acid (2×15 ml), water (2×15 ml), dried overmagnesium sulfate, and reduced in vacuo. Purification by columnchromatography (SiO₂, petroleum:ether (40:60 v/v) with an increasinggradient of ethyl acetate, 0-25%)) gave 0.68 g (80%) of the ketone as ayellow solid.

(c) A mixture of the ketone (1 g, 5.2 mmol), 4-methoxybenzaldehyde(0.612 ml, 5.1 mmol), piperidine (1.14 ml, 11.5 mmol) and glacial aceticacid (0569 ml, 9.9 mmol) in dry ethanol (100 ml) were heated underreflux and water was removed from the reaction by soxhlet extractionover 4A molecular sieves for 50 hours. The solvent was removed in vacuoand the residue was purified by column chromatography (SiO₂,petroleum:ether (40:60 v/v) with an increasing gradient of ethyl acetate(0-25%)). Further purification by recrystallisation from methanol gave0.390 g (24%) of the product as pale yellow crystals.

¹H-NMR (CDCl₃) 7.4 (d, 2H), 7.2 (d, 1H), 6.9 (d, 2H), 6.8 (d, 2H), 6.6(t, 1H), 3.9 (s, 3H), 3.85 (s, 3H), 2.3 (s, 3H). ¹³C NMR (CDCl₃) 188.9,160.5, 144.0, 141.6, 140.9, 134.7, 132.9, 130.5, 128.4, 115.3, 112.7,108.5, 106.0, 104.9, 102.5, 56.7, 54.4. Mass Spectrum m/e (M+1) 313.

Example 6(E)-1-(3-Hydroxy-4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-oneoxime (Compound VII, DMU-153)

To a solution of hydroxylamine hydrochloride (0.5 g) in water (2 ml) wasadded an aqueous solution of sodium hydroxide (10% w/v, 2 ml), followedby the addition of a solution of Compound III (DMU-153, 0.1 g) dissolvedin ethanol (2 ml). The mixture was heated under reflux for 30 min, andthen cooled in an ice bath to give the product as a white crystallinesolid.

Example 7(E)-1-(3-acetoxy-4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one(Compound VIII, DMU-170)

To a stirred solution of Compound III (DMU-153) (0.314 g, 1 mmol) inchloroform (20 ml) was added N-ethyldiisopropylamine (0.171 ml, 1 mmol)and acetyl chloride (0.711 ml, 10 mmol) and the mixture stirred for 24hours at room temperature. The mixture was diluted with water (20 ml)and the organic phase separated and dried over anhydrous sodium sulfate.Evaporation and recrystallisation from methanol afforded the product asa cream solid (0.30 g, 84%).

¹H NMR δ (CDCl₃) 2.34 (3H, s, OCOCH₃), 3.86 (6H, s, OCH₃), 3.88 (3H, s,OCH₃), 6.66 (1H, t, J=2.3 Hz, H-4′), 6.98 (1H, d, J=8.5 Hz, H-5), 7.12(2H, d, J=2.3 Hz, H-2′,6′), 7.30 (1H, d, J=15.6 Hz, CH), 7.38 (1H, d,J=2.2 Hz, H-2), 7.46 (1H, dd, J=2.2, 8.5 Hz, H-6), 7.73 (1H, d, J=15.6Hz, CHCO); ¹³C NMR δ (CDCl₃) 20.56, 55.61, 56.02, 104.96, 106.29,112.38, 120.77, 122.06, 128.04, 128.39, 140.13, 140.34, 143.78, 153.17,160.89, 168.75, 189.85; MS m/z 357 (M+1); IR (KBr) cm⁻¹ 1780, 1650,1590; Anal. Calcd (C₂₀H₂₀O₆): C, 67.41; H, 5.66. Found C, 67.13; H,5.69.

Example 8 Anticancer Activity

Compound III was tested against a number of human tumour cell lines andfound to be a very potent anticancer agent. In particular it has highlypotent anticancer activity against human tumours such as breast tumours,colon tumours, and lung tumours. The results are summarised in Table 1.

TABLE 1 Anticancer Activity of Compound III (results expressed as doserequired for 50% inhibition of tumour cell growth, IC50/μM) Tumour CellsCompound III (DMU-153) IC50/μM Breast MCF-7 0.00065 Colon HCT-1160.00008 Lung A-549 0.08  

Compound IV (DMU-428) was tested against Breast MCF-7 tumour cells, andalso found to be a very potent anticancer agent. The results aresummarised in Table 2.

TABLE 2 Anticancer Activity of Compound IV (results expressed as doserequired for 50% inhibition of tumour cell growth, IC50/μM) Tumour CellsCompound IV (DMU-428) IC50/μM Breast MCF-7 0.0001

The human tumour cell lines were the breast cancer cell line MCF-7, thecolon cancer cell line HCT-116, and the lung cancer cell line A-549. TheMTT assay was used, which exploits the ability of living cells tometabolise the water soluble tetrazolium salt3-[4,5-dimethylthiazol-2yl-2,5-diphenyl tetrazolium bromide (MTT) into awater insoluble formazan precipitate (Carmichael et al, 1987). Thepurple precipitate can then be dissolved in an organic solvent and theoptical density determined as a measure of cell survival. Compounds weretested concurrently for a true comparison.

The compound under study was dissolved in DMSO to yield a 10 mM stocksolution. The final concentration of DMSO (1% max) was found to haveminimal effects on the assay result. Cells were seeded into sterileflat-bottomed 96-well plates at a known initial seeding density (MCF-71.5×10E3, HCT-116 1×10E3, A549 2×10E3). The cells were plated in RPMI1640 medium supplemented with 10% heat-inactivated Foetal Calf Serumsolution. The plates were then incubated for 24 h at 37° C., 5% CO₂ toallow cell adherence. 20 μl of the appropriate drug dilution (fromserial dilutions in medium from 10 mM stock) was added to the wells (togive a final well volume of 200 μl). Plates were returned to theincubator for 96 h, and then 50 μl of MTT was added to each well. Aftera further 4 h incubation, the medium and any unconverted MTT wasaspirated from the wells and the formazan precipitate dissolved by theaddition of 100 μl of DMSO and several minutes agitation. The absorbanceat A₄₅₀ was then recorded on a plate reader, and the results expressedas a % survival of DMSO treated controls. From this data was calculatedthe concentration at which 50% cytotoxicity is observed (IC₅₀).

Compound III exhibits very potent activity against the breast cancercell line MCF-7 with an IC₅₀ of 0.00065 μM. The activity against thecolon tumour cell line HCT-116 is even more impressive with an extremelypotent activity of 0.00008 μM (0.08 nM). The activity against the lungcancer cell line A-549 is also of useful potency at 0.08 μM, since thisis a cell line derived from a non-small cell lung carcinoma (NSCLC)which is refractory to commonly used chemotherapeutic agents.

Compound IV is even more potent than Compound III against the breastcancer cell line MCF-7, with an IC₅₀ of 0.0001 μM.

Example 9 Compounds as Prodrugs

Compound II is metabolised by the cytochrome P-450 enzyme CYP1B1 throughan aromatic hydroxylation reaction to generate compound III, and thuscompound II acts a prodrug which is activated by CYP1B1 to generate thehighly potent anticancer compound III.

A microsomal preparation of human tumour tissue expressing the CYP1B1enzyme was prepared essentially as described by the method of Barrie etal., 1989. The experiment was carried out at 37° C., under yellow light.An array of 1.5 ml centrifuge tubes were set up in a water bathshaker-under aerobic conditions. To each tube was then added 500 μl ofpH 7.6 buffer (0.1 M NaK₂PO₄), followed by NADPH (5 μl of a 25 mM stocksolution). The microsomal preparation (80 μl) was then added and thetubes preincubated for 5 min at 37° C.

The prodrug substrate, compound II, was then added (10 μl of a 5 mMstock solution) and incubated for 1 h at 37° C. After 1 h the tubes weretransferred to an ice/water cooling bath (0° C.). The tubes were thencentrifuged at 15,000 rpm for 30 min. A sample of the supernatant (100μl) was then taken and analysed by HPLC. HPLC conditions: Spherisorb C18(25 cm×4.6 mm id), used without guard column. Flow rate 1 ml/min. Eluent75% 0.1 M KH₂PO₄ and 25% acetonitrile.

The hydroxylated metabolite, compound III, was detected by HPLC, andconfirmed by comparison with the authentic hydroxylated syntheticcompound III. More specifically, compound II,(E)-1-(4-Methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one, wasconverted to the hydroxylated metabolite compound III,(E)-1-(3-Hydroxy-4-methoxyphenyl)-3-(3,5-dimethoxyphenyl)prop-1-en-3-one.

The anticancer activity of Compound II, and the resulting CYP1B1hydroxylated metabolite compound III, were determined using the methodsdescribed above. The results are summarised in Table 3.

TABLE 3 Anticancer Activity of Compound II and its CYP1B1 HydroxylatedMetabolite Compound III (results expressed as dose required for 50%growth inhibition, IC50/μM) Selectivity Compound II Compound III Factor(DMU-120) (DMU-153) (Prodrug/ Tumour IC50/μM IC50/μM Metabolite) BreastMCF-7 0.69 0.00065 1100 Colon HCT-116 1.2 0.00008 15000 Lung A-549 6.60.08 80

Example 10 Anti-Inflammatory Activity

The compounds of the present invention also show growth down-regulatoryeffects on splenocytes. Since splenocytes are involved in inflammation,these compounds are also useful as anti-inflammatory agents.

The anti-inflammatory effects of Compound II (DMU-120) were examined (intriplicate) using a splenocyte anti-proliferation assay.

The splenocyte anti-proliferation assay has been developed to identifycompounds that have useful anti-inflammatory properties for thetreatment of auto-inflammatory diseases such as rheumatoid arthritis.See, for example, Yamashita et al., 1994. This well known assay isdescribed in detail in, for example, Mosmann, 1983. In this assay,splenocyte proliferation is stimulated by the inflammatory responseinducer conconavilin A (Con A). Cell proliferation is monitored bydetecting radiation (counts per minute, cpm) from a radio label(tritiated thymidine) which is incorporated only into proliferatingcells.

The results are summarised in FIG. 1. The compound was assayed as asolution in dimethylsulfoxide (DMSO) as solvent. The solvent control isalso shown for comparison. Other controls are also shown. “ConA” denotesthe signal (cpm) detected for a control where cell proliferation isstimulated by ConA in the absence of a test compound. “Med” denotes thesignal (cpm) detected for the cell culture medium alone. “Bkg” denotesthe background radiation level (cpm).

Compounds that exhibit anti-inflammatory effects at a concentration ofless than 10 μM are considered to be useful therapeutic agents. CompoundII clearly shows anti-inflammatory effects on splenocyte proliferationat concentrations less than 10 μM and therefore exhibits usefulanti-inflammatory properties.

REFERENCES

A number of patents and publications are cited above in order to morefully describe and disclose the invention and the state of the art towhich the invention pertains. Full citations for these references areprovided below. Each of these references is incorporated herein byreference in its entirety into the present disclosure.

Akihiko, 1986, Japanese Patent Publication No. 61-076433, published Apr.18, 1986.

Barrie et al., 1989, J. Steroid Biochem., Vol. 6, pp. 1191-1195.

Berge et al., 1977, “Pharmaceutically Acceptable Salts,” J. Pharm. Sci.,Vol. 66, pp. 1-19.

Berryman et al., 1995, published International Patent Application,publication number WO 95/05376, published Feb. 23, 1995.

Berryman et al., 1997, U.S. Pat. No. 5,691,373, issued Nov. 25, 1997.

Carmichael et al., 1987, “Title,” Cancer Research, Vol. 47, p. 936.

Ducki et al., 1998, “Potent Antimitotic and Cell growth InhibitoryProperties of Substituted Chalcones,” BioMed. Chem. Lett., Vol. 8, pp.1051-1056.

Hiromitsu, 1996, Japanese Patent Publication No 08-188546, publishedJul. 23, 1996.

Mosmann, T., 1983, Journal of Immunological Methods, Vol. 65, pp. 55-63.

Murray et al., 1997, “Title,” Cancer Research, Vol. 57, p. 3026.

Pettit et al, 1995, “Synthesis of Combretastatin prodrugs,” AnticancerDrug Design, Vol. 10, pp. 299-309.

Yamashida, D. S., et al, 1994, Bioorg. Med. Chem. Lett., Vol. 4, pp.325-328.

What is claimed is:
 1. A compound of the formula:

wherein: X is —H, —OH, —OC(═O)R³, —OS(═O)₂OH, or —OP(═O)(OH)₂; Y is —Hor a C₁₋₄alkyl group; Z is —H or —OCH₃; R¹ is —H, a C₁₋₄alkyl group, orC₁₋₄fluoroalkyl group; R² is —H, a C₁₋₄alkyl group, or C₁₋₄fluoroalkylgroup; and, R³ is —H, a C₁₋₆alkyl group, a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀aryl group; or a pharmaceutically acceptable salt, ester, orprotected form thereof; with the proviso that X, Y, Z, R¹, and R² arenot all —H.
 2. A compound according to claim 1, wherein X is —H.
 3. Acompound according to claim 1, wherein X is —OH, —OC(═O)R³, —OS(═O)₂OH,or —OP(═O)(OH)₂.
 4. A compound according to claim 1, wherein X is —OH.5. A compound according to claim 1 wherein Y is —H, —CH₃, or —CH₂CH₃. 6.A compound according to claim 2, wherein Y is —H, —CH₃, or —CH₂CH₃.
 7. Acompound according to claim 4, wherein Y is —H, —CH₃, or —CH₂CH₃.
 8. Acompound according to claim 1, wherein Y is —H.
 9. A compound accordingto claim 2, wherein Y is —H.
 10. A compound according to claim 4,wherein Y is —H.
 11. A compound according to claim 1, wherein Z is —H.12. A compound according to claim 2, wherein Z is —H.
 13. A compoundaccording to claim 4, wherein Y is —H.
 14. A compound according to claim5, wherein Z is —H.
 15. A compound according to claim 6, wherein Z is—H.
 16. A compound according to claim 7, wherein Z is —H.
 17. A compoundaccording to claim 8, wherein Z is —H.
 18. A compound according to claim9, wherein Z is —H.
 19. A compound according to claim 10, wherein Z is—H.
 20. A compound according to claim 1, wherein Z is —OCH₃.
 21. Acompound according to claim 2, wherein Z is —OCH₃.
 22. A compoundaccording to claim 4, wherein Z is —OCH₃.
 23. A compound according toclaim 5, wherein Z is —OCH₃.
 24. A compound according to claim 6,wherein Z is —OCH₃.
 25. A compound according to claim 7, wherein Z is—OCH₃.
 26. A compound according to claim 8, wherein Z is —OCH₃.
 27. Acompound according to claim 9, wherein Z is —OCH₃.
 28. A compoundaccording to claim 10, wherein Z is —OCH₃.
 29. A compound according toclaim 1, wherein R¹ and R² are independently —H, —CH₃, —CH₂CH₃, —CF₃,—CH₂CF₃, or —CF₂CF₃.
 30. A compound according to claim 1, wherein bothR¹ and R² are —H.
 31. A compound according to claim 1, wherein R³ is—CH₃, —CH₂CH₃, —C(CH₃)₃, or -Ph.
 32. A compound according to claim 1,having the formula:


33. A compound according to claim 1, having the formula:


34. A compound according to claim 1, having the formula:


35. A compound according to claim 1, having the formula:


36. A compound according to claim 1, having the formula:


37. A compound according to claim 1, having the formula:


38. A compound according to claim 1, having the formula:


39. A compound according to claim 1, having the formula:


40. A compound according to claim 1, having the formula:


41. A compound according to claim 1, having the formula:


42. A composition comprising a compound according to claim 1 and apharmaceutically acceptable carrier.
 43. A method of treating aproliferative condition in a patient comprising administering to saidpatient a therapeutically-effective amount of a compound having theformula:

wherein: X is —H, —OH, —OC(═O)R³, —OS(═O)₂OH, or —OP(═O)(OH)₂; Y is —Hor a C₁₋₄alkyl group; Z is —H or —OCH₃; R¹ is —H, a C₁₋₄alkyl group, orC₁₋₄fluoroalkyl group; R² is —H, a C₁₋₄alkyl group, or C₁₋₄fluoroalkylgroup; and, R³ is —H, a C₁₋₆alkyl group, a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀aryl group; or a pharmaceutically acceptable salt, ester, orprotected form thereof.
 44. A method according to claim 43, wherein X is—H.
 45. A method according to claim 43, wherein X is —OH, —OC(═O)R³,—OS(═O)₂OH, or —OP(═O)(OH)₂.
 46. A method according to claim 43, whereinX is —OH.
 47. A method according to claim 43, wherein Y is —H.
 48. Amethod according to claim 44, wherein Y is —H.
 49. A method according toclaim 46, wherein Y is —H.
 50. A method according to claim 43, wherein Zis —H.
 51. A method according to claim 44, wherein Z is —H.
 52. A methodaccording to claim 46, wherein Z is —H.
 53. A method according to claim47, wherein Z is —H.
 54. A method according to claim 48, wherein Z is—H.
 55. A method according to claim 49, wherein Z is —H.
 56. A methodaccording to claim 43, wherein Z is —OCH₃.
 57. A method according toclaim 44, wherein Z is —OCH₃.
 58. A method according to claim 46,wherein Z is —OCH₃.
 59. A method according to claim 47, wherein Z is—OCH₃.
 60. A method according to claim 48, wherein Z is —OCH₃.
 61. Amethod according to claim 49, wherein Z is —OCH₃.
 62. A method accordingto claim 43, wherein both R¹ and R² are —H.
 63. A method according toclaim 43, wherein both R³ is —CH₃, —CH₂CH₃, —C(CH₃)₃, or -Ph.
 64. Amethod according to claim 43, wherein the compound has the formula:


65. A method according to claim 43, wherein the compound has theformula:


66. A method according to claim 43, having the formula:


67. A method according to claim 43, having the formula:


68. A method according to claim 43, having the formula:


69. A method according to claim 43, having the formula:


70. A method according to claim 43, having the formula:


71. A method according to claim 43, having the formula:


72. A method according to claim 43, having the formula:


73. A method according to claim 43, having the formula:


74. A method according to claim 43, having the formula:


75. A method according to claim 43, wherein the proliferative conditionis cancer.
 76. A method of prophylactically treating a proliferativecondition in a patient comprising administering to said patient atherapeutically-effective amount of a compound having the formula:

wherein: X is —H, —OH, —OC(═O)R³, —OS(═O)₂OH, or —OP(═O)(OH)₂; Y is —Hor a C₁₋₄alkyl group; Z is —H or —OCH₃; R¹ is —H, a C₁₋₄alkyl group, orC₁₋₄fluoroalkyl group; R² is —H, a C₁₋₄alkyl group, or C₁₋₄fluoroalkylgroup; and, R³ is —H, a C₁₋₆alkyl group, a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀aryl group; or a pharmaceutically acceptable salt, ester, orprotected form thereof.
 77. A method according to claim 76, wherein theproliferative condition is cancer.
 78. A method of treating ainflammatory condition in a patient comprising administering to saidpatient a therapeutically-effective amount of a compound having theformula:

wherein: X is —H, —OH, —OC(═O)R³, —OS(═O)₂OH, or —OP(═O)(OH)₂; Y is —Hor a C₁₋₄alkyl group; Z is —H or —OCH₃; R¹ is —H, a C₁₋₄alkyl group, orC₁₋₄fluoroalkyl group; R² is —H, a C₁₋₄alkyl group, or C₁₋₄fluoroalkylgroup; and, R³ is —H, a C₁₋₆alkyl group, a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀aryl group; or a pharmaceutically acceptable salt, ester, orprotected form thereof.
 79. A method according to claim 78, wherein theinflammatory condition is rheumatoid arthritis, rheumatic fever,osteoarthritis, inflammatory bowel disease, psoriasis, or bronchialasthma.
 80. A method of diagnosis of a patient for the presence oftumour cells expressing the CYP1B1 enzyme, comprising: (a) administeringto the patient a compound having the formula:

wherein: X is —H; Y is —H or a C₁₋₄alkyl group; Z is —H or —OCH₃; R¹ is—H, a C₁₋₄alkyl group, or C₁₋₄fluoroalkyl group; R² is —H, a C₁₋₄alkylgroup, or C₁₋₄fluoroalkyl group; and, R³ is —H, a C₁₋₆alkyl group, aC₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group; or a pharmaceuticallyacceptable salt, ester, or protected form thereof; (b) determining theamount of the corresponding hydroxylated metabolite, wherein X is —OH,which is subsequently produced; and, (c) correlating the amount with thepresence or absence of the tumour cells in the patient.
 81. A compoundof the formula:

wherein: X is —H, —OH, —OC(═O)R³, —OS(═O)₂OH, or —OP(═O)(OH)₂; Y is —Hor a C₁₋₄alkyl group; Z is —H or —OCH₃; R¹ is —H, a C₁₋₄alkyl group, orC₁₋₄fluoroalkyl group; R² is —H, a C₁₋₄alkyl group, or C₁₋₄fluoroalkylgroup; and, R³ is —H, a C₁₋₆alkyl group, a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀aryl group; or a pharmaceutically acceptable salt, ester, orprotected form thereof; with the proviso that X, Y, Z, R¹, and R² arenot all —H and at least two of X, Y and Z are not hydrogen when R¹ andR² are hydrogen.
 82. A compound according to claim 1, wherein Z is —OCH₃and X is —OH, —OC(═O)R³, —OS(═O)₂OH, or —OP(═O)(OH)₂.
 83. A compoundaccording to claim 1, wherein Z is —OCH₃ and Y is a C₁₋₄alkyl group. 84.A method of treating a proliferative condition in a patient comprisingadministering to said patient a therapeutically-effective amount of acompound of claim 81, or a pharmaceutically acceptable salt, ester, orprotected form thereof.
 85. A method of prophylactically treating aproliferative condition in a patient comprising administering to saidpatient a therapeutically-effective amount of a compound of claim 81 ora pharmaceutically acceptable salt, ester, or protected form thereof.86. A method according to claim 85, wherein the proliferative conditionis cancer.
 87. A method of treating a inflammatory condition in apatient comprising administering to said patient atherapeutically-effective amount of a compound of claim 81 or apharmaceutically acceptable salt, ester, or protected form thereof. 88.A method according to claim 87, wherein the inflammatory condition isrheumatoid arthritis, rheumatic fever, osteoarthritis, inflammatorybowel disease, psoriasis, or bronchial asthma.
 89. A method of diagnosisof a patient for the presence of tumour cells expressing the CYP1B1enzyme, comprising: (a) administering to the patient a compound of claim81, or a pharmaceutically acceptable salt, ester, or protected formthereof; (b) determining the amount of the corresponding hydroxylatedmetabolite, wherein X is —OH, which is subsequently produced; and, (c)correlating the amount with the presence or absence of the tumour cellsin the patient.